This invention relates generally to novel mutant hemoglobins and more particularly relates to recombinant mutant hemoglobins xe2x80x9crHb (xcex2N108Q)xe2x80x9d (alternative designation xe2x80x9crHb (xcex2108Asnxe2x86x92G1n)xe2x80x9d) and xe2x80x9crHb (xcex2L105W)xe2x80x9d (alternative designation xe2x80x9crHb (xcex2L105Leuxe2x86x92Trpxe2x80x9d) that possess low oxygen affinity, and high cooperativity in oxygen binding. In particular, rHb (xcex2N108Q) exhibits increased resistance to autoxidation as compared to other known low oxygen affinity mutants. This invention further relates to the preparation of mutant hemoglobins using recombinant DNA technology that are useful as substitutes for red blood cells and for hemoglobin-based therapeutics.
The prevalence of infectious agents such as HIV and hepatitis in red blood cells of human blood products coupled with blood shortages from lack of suitable donors has led to great interest in the development of red blood cell substitutes, particularly human hemoglobin (xe2x80x9cHbxe2x80x9d) and its derivatives. Hemoglobin-based oxygen carriers are potential sources of blood substitutes during emergency medical situations. See for example, Winslow, R. M., et al. Hemoglobin-Based Red Cell Substitutes, Johns Hopkins University Press, Baltimore (1992) (hereinafter xe2x80x9cWinslow, et al. (1992)xe2x80x9d), the disclosure of which is incorporated herein by reference.
Hemoglobin is the oxygen-carrying component of blood, circulated through the blood stream inside erythrocytes (red blood cells). Human normal adult hemoglobin (xe2x80x9cHb Axe2x80x9d) is a tetrameric protein with a molecular weight of about 64,500 containing two identical a chains having 141 amino acid residues each and two identical xcex2 chains having 146 amino acid residues each, with each also bearing prosthetic groups known as hemes. The erythrocytes help maintain hemoglobin in its reduced, functional form. The heme-iron atom is susceptible to oxidation, but may be reduced again by one of two systems within the erythrocyte, the cytochrome b5, and glutathione reduction systems. For a review on hemoglobin, see, Dickerson, R. E., et al. Hemoglobin: Structure, Function, Evolution, and Pathology, p. 22-24, Benjamin/Cummings, Menlo Park, Calif. (1983) (hereinafter xe2x80x9cDickerson, et al. (1983)xe2x80x9d), the disclosure of which is incorporated herein by reference.
The oxygenation process of Hb A is cooperative, i.e., the binding of the first oxygen molecule enhances the binding of the second, third, and fourth oxygen molecules. The oxygenation process is also regulated by interactions between individual amino acid residues and various solutes, known as heterotropic allosteric effectors. These effectors include ions or molecules such as hydrogen ion, chloride, carbon dioxide, inorganic phosphate, and organic polyanions, such as 2,3-bisphosphoglycerate (xe2x80x9c2,3-BPGxe2x80x9d) and inositol hexaphosphate (xe2x80x9cIHPxe2x80x9d).
Hemoglobin is able to alter its oxygen affinity, thereby increasing the efficiency of oxygen transport in the body, due to its dependence on the allosteric effector 2,3-BPG. 2,3-BPG is present within erythrocytes at a concentration that allows hemoglobin to release bound oxygen to tissues. In the absence of 2,3-BPG, hemoglobin binds oxygen very tightly and does not readily release its bound oxygen. The Hub A molecule alone, were it to be introduced into a subject, would not be able to properly allow oxygen to be delivered to tissues in the body due to a lack of 2,3-BPG, which lowers the oxygen affinity of Hb, in the blood plasma. See, Winslow, et al. (1992). Any Hbs designed to be functional as Hb-based oxygen carriers or hemoglobin therapeutics should be able to deliver oxygen efficiently, i.e., they should load and unload cooperatively as Hb A does inside red blood cells.
The use of cell-free solutions of hemoglobin as a potential oxygen-carrying red cell substitute has been investigated for a long time. See, for example Mulder, A. G., et al., J. Cell Comp. Physiol. 5:383 (1934), the disclosure of which is incorporated herein by reference. However, the use of unmodified cell-free human hemoglobin purified from red blood cells suffers from several limitations in addition to contamination and supply limitations noted above, namely, an increase in oxygen affinity due to loss of allosteric effectors, such as 2,3-BPG, and dissociation of Hb tetramers into xcex1xcex2 dimers which are cleared by renal filtration and which can cause long-term kidney damage. See, for example Bunn, H. F., et al. J. Exp. Med. 129:909 (1969), the disclosure of which is incorporated herein by reference.
Human globins and hemoglobins have been expressed in the following: transgenic mice, see, for example, Chada, K., et al., Nature (London) 314:377 (1985) and Townes, T. M., et al. EMBO J. 4:1715 (1985), transgenic swine as described by Swanson, M. E., et al. Bio/Technology 10:557 (1992), insect cell cultures as reported by Groebe, D. R., et al., Protein Expression and Purification 3:134 (1992), yeast as described by Wagenbach, M., et al. Bio/Technology 9:57 (1991) and DeLiano, J. J., et al. Proc. Natl. Acad. Sci. USA 90:918 (1993), and Escherichia coli (xe2x80x9cE. colixe2x80x9d) as described by Hoffman, S. J., et al. Proc. Natl. Acad. Sci. USA 87:8521 (1990), Hernan, R. A., et al. Biochemistry 31:8619 (1992), and Shen, T. -J., et al. Proc. Natl. Acad. Sci. USA 90:8108 (1993) (hereinafter xe2x80x9cShen, et al. (1993)xe2x80x9d), all the disclosures of which are incorporated herein by reference. In many respects, the E. coli system is the best choice for such purposes because of its high expression efficiency and the ease of performing site-directed mutagenesis.
The natural N-terminal valine residues of Hb A are known to play important roles in regulating oxygen affinity, the Bohr effect, and interactions with allosteric effectors and anions as reported by Bunn, H. F., et al. eds. Hemoglobin: Molecular, Genetic and Clinical Aspects (W. B. Saunders, Co., Philadelphia, Pa.) pp. 37-60 (1986) (hereinafter xe2x80x9cBunn, et al. (1986)xe2x80x9d), the disclosure of which is incorporated herein by reference. The extra methionine can alter the N-terminal conformation of the Hb molecule as reported by Kavanaugh, J. S., et al. Biochemistry 31:8640 (1992), the disclosure of which is incorporated herein by reference. Hence, the oxygenation properties of Hb depend on the integrity of the N-terminal residue thereby mandating the removal of the extra methionine residues from the N-termini of both the xcex1- and xcex2-globins of the expressed Hb before the E. coli system can be used effectively for the production of desired unmodified and mutant Hbs.
The cooperative oxygenation of Hb, as measured by the Hill coefficient (xe2x80x9cnmaxxe2x80x9d) is a convenient measure of its oxygenation properties. See, Dickerson, et al. (1983). Hb A has an nmax value of approximately 3 in its binding with O2 under usual experimental conditions. Human abnormal Hbs with amino acid substitutions in the xcex11xcex22 (or xcex12xcex21) subunit interface generally result in high oxygen affinity and reduced cooperativity in O2 binding compared to Hb A. See, for example, Dickerson, et al. (1983); Bunn, at al (1986) and Perutz, M. F. at al. Mechanisms of Cooperativity and Allosteric Regulation in Proteins pp. 19-29, Cambridge University Press (1990), the disclosure of which is incorporated herein by reference.
Hb A in its oxy form (Hb A with oxygen molecules) has a characteristic hydrogen bond between xcex194Asp and xcex2102Asn in the xcex11xcex22 subunit interface as reported by Shaanan, B., et al. J. Mol. Biol. 171:31 (1983), the disclosure of which is incorporated herein by reference (hereinafter xe2x80x9cShaanan, et al. (1983)xe2x80x9d). Human Hbs with an amino acid substitution at either the xcex194Asp position such as Hb Titusville (xcex194Aspxe2x86x92Asn) (Schneider, R. G., et al. Biochim. Biophys. Acta. 400:365 (1975), the disclosure of which is incorporated herein by reference) or the xcex2102Asn position such as Hb Kansas (xcex2102Asnxe2x86x92Thr) (Bonaventura, J., et al. J. Biol. Chem. 243:980 (1968), the disclosure of which is incorporated herein by reference), as well as others with mutations in the xcex11xcex22 subunit interface, exhibit very low oxygen affinity. However, all these Hb mutants which directly disrupt the hydrogen bond between xcex194Asp and xcex2102Asn in the oxy form of Hb show greatly reduced cooperativity in the binding of oxygen and additionally dissociate easily into dimers when in the ligated state.
It has also been shown that during the transition from the deoxy-to the oxy-state, the xcex11xcex22 subunit of Hb A undergoes a sliding movement, while the xcex11xcex21 subunit interface remains nearly unchanged (See, Perutz, M. F. Nature 228: 726 (1970) (xe2x80x9cPerutz (1970)xe2x80x9d); Baldwin, J. M., et al. J. Mol. Biol. 129: 175 (1979); Baldwin, J. M., J. Mol. Biol. 136: 103 (1980); Shaanan, et al. (1983); and Fermi, G., et al. J. Mol. Biol. 175: 159 (1984), (xe2x80x9cFermi, et al., (1984)xe2x80x9d), the disclosures of which are incorporated herein by reference. There are specific hydrogen bonds, salt bridges, and non-covalent interactions that characterize both subunit interfaces. The Hb molecule also has a lower oxygen affinity in the deoxy quaternary structure (T-structure) than in the oxy quaternary structure (R-structure) See, Dickerson, et al. (1983).
Low oxygen affinity human mutant Hbs which do not involve either xcex194Asp or xcex2102Asn also exist. For example, Hb Presbyterian (xcex2108Asnxe2x86x92Lys) (Moo-Penn, W. F., et al. FEBS Lett. 92:53 (1978) and O""Donnell, J. K., et al. J. Biol. Chem. 269:27692 (1994) (hereinafter xe2x80x9cO""Donnell, et al. (1994)xe2x80x9d); Hb Yoshizuka (xcex2108Asnxe2x86x92Asp), O""Donnell, et al. (1994) and recombinant Hb Mequon (xcex241Phexe2x86x92Tyr) (Baudin, V., et al. Biochim. Biophys. Acta. 1159:223 (1992), the disclosures of which are incorporated herein by reference, all exhibit low oxygen affinity compared to Hb A, but they all exhibit a variable amount of cooperativity as measured by the Hill coefficient, with n varying from 1.8 to 2.9. Tsai, C. -H., et al. Biochemistry 38:8751 (1999) (hereinafter, xe2x80x9cTsai, et al. (1999)xe2x80x9d) report Hb (xcex196Valxe2x86x92Trp, xcex2108Asnxe2x86x92Lys) which has low oxygen affinity and a greater tendency to switch to the T quaternary structure. Jeong, S. T., et al., Biochemistry 38:13433 (1999) (hereinafter, xe2x80x9cJeong, et al. (1999)xe2x80x9d) report that Hb (xcex129Leuxe2x86x92Phe, xcex196Valxe2x86x92Trp, xcex2108Asnxe2x86x92Lys) exhibits low oxygen affinity and high cooperativity combined with resistance to autoxidation.
Shen, et al. (1993) and U.S. Pat. No. 5,753,465, the disclosures of which are incorporated herein by reference, describe an E. coli expression plasmid (pHE2) in which synthetic human xcex1- and xcex2-globin genes are coexpressed with the E. coli methionine aminopeptidase gene under the control of separate tac promotors. E. coli cells transformed with this plasmid express recombinant Hb A (hereinafter xe2x80x9crHb Axe2x80x9d) from which the N-terminal methionines have been effectively cleaved by the coexpressed methionine aminopeptidase. The resulting rHb A which lacks an N-terminal methionine is identical to the native Hb A in a number of structural and functional properties.
Kim, H. -W., et al. Proc. Natl. Acad. Sci. USA 91:11547 (1994) (hereinafter xe2x80x9cKim, et al. (1994)xe2x80x9d), and U.S. Pat. No. 5,843,888, the disclosures of which are incorporated herein by reference, describe a non-naturally occurring mutant hemoglobin (rHb(xcex196Valxe2x86x92Trp) (alternative designation xe2x80x9crHb (xcex196W)xe2x80x9d) that has a lower oxygen affinity than that of native hemoglobin, but high cooperativity in oxygen binding.
There remains a need, however, for additional mutant hemoglobin species that can be used as a component of a hemoglobin-based blood substitute or therapeutic agent. Of particular interest is a mutant hemoglobin that possesses low oxygen affinity, high cooperativity in oxygen binding, and increased stability against autoxidation. There is a further need for such a hemoglobin produced by recombinant methods and an efficient expression system for producing such a mutant hemoglobin in high yield, especially for use in a blood substitute product or hemoglobin therapeutics.
Accordingly, it is a primary object of the present invention to provide mutant human hemoglobins with low oxygen affinity and high cooperativity in oxygen binding.
Another object of the present invention is to provide mutant hemoglobins with low oxygen affinity, high cooperativity in oxygen binding, and increased stability against autoxidation.
Another object of the present invention is to provide non-naturally occurring mutant human hemoglobins with low oxygen affinity and high cooperativity in oxygen binding.
Another object of the present invention is to provide non-naturally occurring mutant human hemoglobins with low oxygen affinity, high cooperativity in oxygen binding, and increased stability against autoxidation.
Another object of the present invention is to provide non-naturally occurring mutant human hemoglobins with low oxygen affinity, high cooperativity in oxygen binding, and preferably with stability against autoxidation that are produced artificially, preferably by recombinant means, and that have the correct heme conformation.
Another object of the present invention is to provide mutant hemoglobins that in a cell-free environment have similar oxygen binding properties as those of human normal adult hemoglobin in red blood cells.
Yet another object of the present invention is to provide mutant hemoglobins with low oxygen affinity and high cooperativity in oxygen binding in which the T-structure is stabilized while the R-structure is undisturbed.
Still another object of the present invention is to provide artificial hemoglobins for use as a hemoglobin-based oxygen carrier/red blood substitute or therapeutic agent.
These and other objects of the present invention are achieved by one or more of the following embodiments.
In one aspect, the invention features a non-naturally occurring mutant human hemoglobin wherein the asparagine residue at position 108 of the xcex2-chains is replaced by a glutamine residue.
In a preferred embodiment, the hemoglobin possesses low oxygen affinity as compared to human normal adult hemoglobin, high cooperativity in oxygen binding, increased stability against autoxidation, and is produced recombinantly.
In another aspect, the invention features an artificial mutant hemoglobin which in a cell-free enviornment has oxygen binding properties comparable to those of human normal adult hemoglobin in red blood cells wherein said hemoglobin contains a mutation such that the asparagine residue at position 108 of the xcex2-chains is glutamine.
A non-naturally occurring low oxygen affinity mutant hemoglobin with increased stability against autoxidation that has oxygen binding properties comparable to those of human normal adult hemoglobin in the presence of the allosteric effector 2,3-bisphosphoglycerate, wherein the asparagine residue at position 108 of each of the xcex2-chains is replaced by a glutamine residue.
In yet another aspect, the invention features a non-naturally occurring mutant human hemoglobin wherein the leucine residue at position 105 of the xcex2-chains is replaced by a tryptophan residue.
In a preferred embodiment, the hemoglobin possesses low oxygen affinity as compared to human normal adult hemoglobin, high cooperativity in oxygen binding, and is produced recombinantly.
In another aspect, the invention features an artificial mutant hemoglobin which in a cell-free environment has oxygen binding properties comparable to those of human normal adult hemoglobin in red blood cells wherein said hemoglobin contains a mutation such that the leucine residue at position 105 of the xcex2-chains is tryptophan.
A non-naturally occurring low oxygen affinity mutant hemoglobin that has oxygen binding properties comparable to those of human normal adult hemoglobin in the presence of the allosteric effector 2,3-bisphosphoglycerate, wherein the leucine residue at position 105 of each of the xcex2-chains is replaced by a tryptophan residue.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiment, and from the claims.